Gene transfer technologies aim to express genes in different cell types in a stable and/or regulated fashion and without affecting physiological expression of the target cell. This technology has demonstrated to be of crucial importance for discovery, protein production and therapeutic use (gene therapy). However, gene transfer vectors (the tools used to transfer the gene into the cells) are confronted with several obstacles such as:                Gene silencing, most vectors loose their expression due to epigenetic modifications.        Variegation. The expression patterns of integrative vectors such as retrovirus vary depending on the site of integration, making it difficult to control the expression of the transgene.        Finally, the integrative vectors affect the expression pattern of the target cell due to the presence of several elements (enhancers, cryptic splice donor and acceptors, poliadenilation sites) that influence the normal expression of genes located near the integration sites.        
Thus, gene transfer vectors are hampered with problems at the time of expressing a transgene in a target cell in a stable manner. In this sense, the gene transfer vector backbone seems to be the main player determining stability of transgene expression on target cells. In this regard, some vectors are very prompt to gene silencing due to the presence of viral promoters and/or enhancers. In retroviral vectors, the same elements are also responsible for genotoxicity, mainly due to the activation of oncogenes. In addition, the target cell will also dictate the stability of transgene expression and the potential genotoxicity of the integrative vector. In general, transgene expression on stem and primary cells are more prompt to gene silencing than differentiated and/or immortalized cell lines. In terms of vector genotoxicity, stem cells are the only target cells where serious advert effects (cell transformation) have been observed. Therefore, stable gene transfer in stem cells has been hampered by gene silencing and/or genotoxicity due to vector integration.
There are several ways to reduce silencing and genotoxicity of gene transfer vectors. As already mentioned, vector backbone is a main determinant in this phenomena. In this sense, it has been demonstrated that lentiviral vectors have a safer integration profile compared to gammaretroviral vectors. In the same direction, physiological promoters with weak enhancers are less prompt to oncogen activation that strong viral promoters/enhancers. All these studies point to lentiviruses as the vectors of choice for gene transfer when sustain transgene expression is required. Still, lentiviral vectors do integrate within active genes and this could affect the expression pattern of the target cell. In addition, the expression profile of the lentiviral vectors can also be affected by the integration site.
In order to avoid the deleterious effects of lentiviral vector over the host chromatin and viceversa, different groups have included chromatin insulators (i.e. cHS4) and scaffold (matrix) attachment regions (M/SAR) into the lentiviral backbones. Chromatin insulators form expression boundaries that can have two different activities: 1—Enhancer-blocking, reducing interferences between promoters and enhancers located at different sides of the insulator and 2—barrier activity, preventing gene silencing. S/MAR elements bind to the nuclear matrix and is postulated that this binding defines boundaries of independent chromatin domains that can enhance and/or protect gene expression.
The 1.2 kb chromatin insulator from the chicken β-globin locus control region hypersensitive site 4(cHS4) has been the most widely use insulator in retrovirus vectors. The cHS4 is one of the few insulators that have both, enhancer blocking and barrier activity. When incorporated into the LTR of retroviral vectors, the cHS4 insulator provides uniform gene expression thanks to the enhancer-blocking activity. cHS4 Insulated gamma-retrovirus vectors where also able to avoid gene silencing and to decrease genotoxicity by reducing the activation of oncogenes. However, the incorporation of a large 1.2 kb cHS4 into the retroviral LTR causes a drastic reduction in vector titer.
As an alternative to chromatin insulators, some authors have included S/MAR elements into retroviral vectors either alone or in combination. Insertion of the IFN-SAR into gammaretroviral and lentiviral vectors resulted in improved transgene expression.
However, as discussed previously, transgene silencing and genotoxycity is highly dependent on vector backbone and cell type. Indeed, different (sometimes disappointing) results were obtained when the same insulators were used in different vector backbones or when the same insulated vectors were used in different cell types. For example, initial studies found that gammaretroviral flanked with the cHS4 only prevented silencing in about 30%-70% of the time depending on the expression casset. Especially disappointing were the studies targeting human stem cells, where the beneficial effects can be less obvious.
Therefore, there is a need for more efficient barrier insulators to avoid vector silencing in the setting of retroviral vectors.